Pathogenic bacteria must withstand extracytoplasmic stress, or perturbation of the cell envelope and periplasm, to survive in the host environment. An alternative sigma factor called CE (SigmaE, RpoE) controls extracytoplasmic stress responses in Salmonella enterica sv. Typhimurium and other bacteria. We have demonstrated that CE is required for Salmonella virulence and resistance to killing by macrophages and antimicrobial peptides. During the last project period, we have discovered: (a) a novel mechanism of CE activation by acid pH important for survival in macrophages, (b) two new CE-regulated virulence genes encoding a peptidyl-prolyl isomerase and a periplasmic protein of unknown function, and (c) unanticipated interactions linking CE to other stress regulatory networks. The central hypothesis of this proposal is that CE acts in concert with the Rcs, Psp, Cpx and Bae systems to form an integrated regulatory network that defends bacteria from cell envelope damage and maintains pathways of energy generation essential for cellular homeostasis. The specific aims are to explore the mechanisms underlying our recent findings: 1. Analyze the mechanism by which CE is activated by acid pH. Biochemical and genetic approaches will be used to examine the effects of pH on the conformation of the periplasmic RseA domain and its function in the CE regulatory cascade. 2. Determine the mechanism by which the peptidyl-prolyl isomerase SlpA and the periplasmic protein YggN promote bacterial virulence. Proteomic and phenotypic assays will explore the contributions of SlpA and YggN to Salmonella pathogenesis. 3. Investigate the mechanisms by which the Psp and Rcs systems preserve cell survival under stress conditions. A range of functional and biochemical studies will assess the central role of proton motive force and its preservation by extracytoplasmic stress responses. These studies will provide important new insights into conserved mechanisms of bacterial pathogenesis that have broad biological implications.